Peptides molecules represent valuable tools for investigating biological systems, studying the binding and activity properties of biomolecules (e.g., enzymes, cell receptors, antibodies, kinases), exploring the etiopathological causes of diseases, and for validating pharmacological targets. Peptides are also attractive ligands for targeting protein-protein interactions and modulating the function of biological molecules such as enzymes and nucleic acids. The synthesis of combinatorial libraries of small peptides followed by screening of these chemical libraries in biological assays can enable the identification of compounds that exhibit a variety of biological and pharmacological properties. Bioactive peptides identified in this manner can constitute valuable lead compounds or facilitate the development of lead compounds towards the discovery of new drugs.
While many peptides exhibit interesting biological activity, linear peptides do not generally represent suitable pharmacological agents as they are generally only poorly adsorbed, do not cross biological membranes readily, and are prone to proteolytic degradation. In addition, linear peptides fail to bind proteins that recognize discontinuous epitopes. The use of molecular constraints to restrict the conformational freedom of the molecule backbone can be used to overcome these limitations. In many cases, conformationally constrained peptides exhibit enhanced enzymatic stability (Fairlie, Tyndall et al. 2000; Wang, Liao et al. 2005), membrane permeability (Walensky, Kung et al. 2004; Rezai, Bock et al. 2006; Rezai, Yu et al. 2006), and protein binding affinity (Tang, Yuan et al. 1999; Dias, Fasan et al. 2006) and selectivity (Henchey, Porter et al. 2010), compared to their linear counterparts. Constraints that lock-in the active conformation of a peptide molecule can result in increased affinity due to the reduced conformational entropy loss upon binding to the receptor. Many bioactive and therapeutically relevant peptides isolated from natural sources occur indeed in cyclized form or contain intramolecular bridges that reduce the conformational flexibility of these molecules (e.g., immunosuppressant cyclosporin A, antitumor dolastatin 3 and diazonamide A, anti-HIV luzopeptin E2, and the antimicrobial vancomycin). Since macrocyclic peptides constitute promising molecular scaffolds for the development of bioactive compounds and therapeutic agents (Katsara, Tselios et al. 2006; Driggers, Hale et al. 2008; Obrecht, Robinson et al. 2009; Mars ault and Peterson 2011), methods for generating macrocyclic peptides and combinatorial libraries thereof, are of high synthetic value and practical utility, in particular in the context of drug discovery.
While cyclic peptides can be prepared synthetically via a variety of known methods (White and Yudin 2011), the possibility to generate macrocyclic peptides starting from genetically encoded polypeptide precursors offers several advantages (Frost, Smith et al. 2013; Smith, Frost et al. 2013). Among these, there are: (a) the high combinatorial potential inherent to the ribosomal synthesis of genetically encoded polypeptides, which can enable the production of very large collections of peptide sequences (108-1010 members or higher) in a cost- and time-effective manner; (b) the possibility to link these peptide libraries to powerful, high-throughput screening platforms such as phage display, mRNA display, or yeast display, in order to identify peptide ligands with the desired property (e.g., high binding affinity toward a target protein); (c) the ease by which these chemical libraries can be deconvoluted in order to identify the library members of interest (i.e., via sequencing of the peptide-encoding DNA or RNA sequence).
Various methods have been developed for producing biological libraries of conformationally constrained peptides (Frost, Smith et al. 2013; Smith, Frost et al. 2013). For example, libraries of disulfide-constrained cyclic peptides have been prepared using phage display and fusing randomized polypeptide sequences flanked by two cysteines to a phage particle as described, e.g., in U.S. Pat. No. 7,235,626. Disulfide bridges are however potentially reactive and this chemical linkage is unstable under reducing conditions or in a reductive environment such as the intracellular milieu. Alternatively, ribosomally produced peptides have also been constrained through the use of cysteine- or amine-reactive cross-linking agents (Millward, Takahashi et al. 2005; Seebeck and Szostak 2006; Heinis, Rutherford et al. 2009; Schlippe, Hartman et al. 2012). A drawback of these methods is the risk of producing multiple undesired products via reaction of the cross-linking agents with multiple sites within the randomized peptide sequence or the carrier protein in a display system. In addition, these methods do not allow for the formation of macrocyclic peptides inside the polypeptide-producing cell host. Other methods have been described that are useful for preparing head-to-tail cyclic peptides by using natural (i.e., naturally occurring) or engineered (i.e., non-naturally occurring, artificial or synthetic) split inteins, as described in U.S. Pat. Nos. 7,354,756, 7,252,952 and 7,105,341. An advantage of these strategies is the possibility to couple the intracellular formation of cyclic peptide libraries with an cell-based reporter or selection system, which can facilitate the identification of functional peptide ligands (Horswill, Savinov et al. 2004; Cheng, Naumann et al. 2007; Naumann, Tavassoli et al. 2008; Young, Young et al. 2011). However, the peptide cyclization efficiency was found to be highly dependent on the peptide sequence (Scott, Abel-Santos et al. 2001). In addition, only head-to-tail cyclic peptides can be obtained through these strategies, which limits the extent of structural diversity of the ligand libraries generated through these methods. Finally, methods have also been reported for generating cyclic peptides through the enzymatic modification of linear peptide precursors (Hamamoto, Sisido et al. 2011; Touati, Angelini et al. 2011). However, the need for exogenous reagents and/or enzyme catalysts for mediating peptide cyclization and, in some cases, moderate cyclization efficiency limit the scope and utility of these approaches toward the generation and screening of cyclic peptide libraries.
Efficient and versatile methods for generating macrocyclic peptides from ribosomally produced polypeptides would thus be highly desirable in the art. The methods and compositions described herein provide a solution to this need, enabling the ribosomal synthesis of cyclic peptides in vitro (i.e., in a cell-free system) and in vivo (i.e., inside a cell or on a surface of a cell) and in various ‘configurations’, namely in the form of macrocyclic peptides, lariat-shaped peptides, or as cyclic peptides fused to a N-terminus or C-terminus of a protein of interest, such as a carrier protein of a display system.
Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention.